Malassezia restricta-mediated Lipoperoxidation: A Novel Trigger in Dandruff.

Dandruff is a common scalp disorder with multiple microbial and host-related factors contributing to its aetiology, including alterations in scalp sebum. Despite existing evidence that the yeast Malassezia restricta plays a key role in the onset of dandruff, the interplay of these factors is poorly understood. Recently, squalene monohydroperoxide and malondialdehyde were established as biomarkers of dandruff-afflicted scalp, highlighting the role of sebum lipoperoxidation in the triggering and maintenance of dandruff, although its mechanism of action is unknown. The current study provides evidence that M. restricta mediates sebum peroxidation, leading to production of squalene monohydroperoxide and malondialdehyde. Furthermore, in vitro data show that these lipoperoxidation products act on epidermal cells and alter the skin barrier. These results support the role of Malassezia restricta-induced lipoperoxides as triggers of dandruff, which suggests that blocking their production could be a novel anti-dandruff treatment approach.

Evidence for the presence of a human saprophytic oral bacterium, Mycoplasma faucium, in the skin lesions of a psoriatic patient.

Psoriasis is currently regarded a mixed autoimmune and autoinflammatory disease. This report describes for the first time the detection of a saprophytic, human oral Tenericutes species, Mycoplasma faucium, in the skin lesions of a patient presenting initially guttate and later plaque psoriasis. An unusual finding in standard histopathology investigation consisted of round and oval thinly stained or unstained, possibly intracellular structures, apparently directly pressing on keratinocyte nuclei of the psoriatic stratum spinosum. In ultrastructural study, wall-less bacteria were present intracellularly in the keratinocytes, mainly of the psoriatic stratum spinosum, and extracellularly in the upper dermis of the psoriatic lesions. M. faucium was consistently detected and identified in the psoriatic skin by general Tenericutes polymerase chain reaction and sequencing in two biopsies performed 31 months apart. This case raises new questions concerning the pathogenesis of psoriasis and its accepted autoimmune/autoinflammatory nature.

Genotypes and population genetics of cryptococcus neoformans and cryptococcus gattii species complexes in Europe and the mediterranean area.

A total of 476 European isolates (310 Cryptococcus neoformans var. grubii, 150 C. neoformans var. neoformans, and 16 C. gattii species complex) from both clinical and environmental sources were analyzed by multi-locus sequence typing. Phylogenetic and population genetic analyses were performed. Sequence analysis identified 74 sequence types among C. neoformans var. neoformans (VNIV), 65 among C. neoformans var. grubii (56 VNI, 8 VNII, 1 VNB), and 5 among the C. gattii species complex (4 VGI and 1 VGIV) isolates. ST23 was the most frequent genotype (22%) among VNI isolates which were mostly grouped in a large clonal cluster including 50% of isolates. Among VNIV isolates, a predominant genotype was not identified. A high percentage of autochthonous STs were identified in both VNI (71%) and VNIV (96%) group of isolates. The 16 European C. gattii species complex isolates analyzed in the present study originated all from the environment and all belonged to a large cluster endemic in the Mediterranean area. Population genetic analysis confirmed that VNI group of isolates were characterized by low variability and clonal expansion while VNIV by a higher variability and a number of recombination events. However, when VNI and VNIV environmental isolates were compared, they showed a similar population structure with a high percentage of shared mutations and the absence of fixed mutations. Also linkage disequilibrium analysis reveals differences between clinical and environmental isolates showing a key role of PLB1 allele combinations in host infection as well as the key role of LAC1 allele combinations for survival of the fungus in the environment. The present study shows that genetic comparison of clinical and environmental isolates represents a first step to understand the genetic characteristics that cause the shift of some genotypes from a saprophytic to a parasitic life style.

The lipid profile of three Malassezia species assessed by Raman spectroscopy and discriminant analysis.

Malassezia yeasts constitute the major eukaryotic cutaneous flora of homoeothermic vertebrates. These lipophilic yeasts are able to cause, trigger, or aggravate common skin diseases under favorable conditions. Species identification and subspecies differentiation is currently based on morphological characteristics, lipid assimilation profile, and molecular tests. Mass spectrometry has been also reported as a reliable, yet costly and labor-intensive, method to classify Malassezia yeasts. Here, we introduce Raman spectroscopy as a new molecular technique able to differentiate three phylogenetically close Malassezia species (M.globosa, M.pachydermatis, and M.sympodialis) by examining their lipid metabolic profile. Using Raman spectroscopy, lipid fingerprints of Malassezia cultures on Leeming-Notman agar, were analyzed by spectral bands assignment and partial least squares discriminant analysis. Our results demonstrate differential utilization of lipid supplements among these three species and the ability of Raman spectroscopy to rapidly and accurately discriminate them by predictive modelling.

Antifungal activity of selected Malassezia indolic compounds detected in culture.

BACKGROUND: Malassezia yeasts produce bioactive indolic substances when grown on L-tryptophan agar. A panel of these substances was tested against commensal and opportunistic fungi, the Minimum Inhibitory Concentration (MIC) was determined and the potential for in loco antifungal activity on the skin was assessed. MATERIALS AND METHODS: Eight indoles were included (malassezin, pityriacitrin, indirubin, indolo[3,2-b]carbazole, 6-formylindolo[3,2-b]carbazole, tryptanthrin, 6-hydroxymethylindolo[3,2-b]carbazole and 6-methylindolo[3,2-b]carbazole) and were tested against 40 fungal strains [yeasts: Malassezia spp.(N = 9); Cryptococcus spp.(N = 10); Candida spp.(N = 7); Yarrowia lipolytica(N = 1); Exophialla dermatitidis (N = 2); moulds: Aspergillus spp.(N = 7); Fusarium spp.(N = 2); Rhizopus oryzae(N = 2)]. The concentration of 5/8 of the tested indoles on diseased skin was calculated from published data. Kruskal-Wallis and Mann-Whitney U tests were employed for group susceptibility evaluation in 33 strains. RESULTS: The MIC range was 0.125-32 µg/mL, and the median log2 MIC was four. Indirubin was the most potent antifungal agent and differed significantly from the others. The highest median MIC was found for FICZ. Malassezia with Candida strains were more susceptible compared to Cryptococcus and Aspergillus, and this inhibitory activity was predicted to be valid also on human skin. CONCLUSIONS: Malassezia yeasts produce indolic species that inhibit an array of clinically significant yeasts and moulds.

Variation of cultured skin microbiota in mothers and their infants during the first year postpartum.

BACKGROUND/OBJECTIVES: The establishment of newborn skin flora depends on the ongoing skin maturation and the existence of potential microbial colonizers within the environment of the infant during a period of intense mother-infant physical interaction. This longitudinal study assessed culturable skin bacteria in the mother-infant dyad during the first year of life. METHODS: A total of 17 mother-infant dyads were swabbed within 24 hours postpartum and at 3, 6, 9, and 12 months. Skin swabbing was performed on two anatomical areas per individual (mothers: chest-abdomen; infants: forehead-buttocks) and were incubated in five different solid culture media to optimize yield. Isolated bacterial species were identified to genus or species level using the API system (BioMeriéux, Marcy l'Etoile, France). RESULTS: A total of 444 microbial strains were isolated belonging to 22 genera: 6 "frequent" (isolated from > 5% samples: S aureus, Proteus, Klebsiella, Pseudomonas, Enterobacter, and Enterococcus) and 16 "infrequent." Isolated genera per individual peaked at 6 months postpartum for mothers and infants (P < 0.05). Enterobacter, Enterococcus, Klebsiella, and Pseudomonas isolation rates varied significantly as a function of sampling time contrary to the rather constant isolation rates of Proteus and S aureus. The rates of concordant isolation of the same microbial species within the mother-infant dyad tended to drop from birth to the end of the first year postpartum. CONCLUSIONS: Distinct variations in the isolation rates of skin commensals from specific anatomical sites of the mother-infant dyad indicate bidirectional microbial transmission. Increasing skin flora individuality of the growing infant was recorded, manifested by declining rates of concordant isolation of the same microbial species from mother and her infant.

Isolation of Candida auris from cystic fibrosis patient, Greece, April 2019.

We report the first isolation of Candida auris in Greece from a sputum culture of a cystic fibrosis patient in their 20s under posaconazole treatment. The pathogen was identified as C. duobushaemulonii by VITEK2YST, but as C. auris by MALDI-TOF MS. This case underscores the need for species-level identification of all non-albicans Candida (NAC) isolates from cystic fibrosis patients and patients with predisposing factors to fungal infection.

Nail Raman spectroscopy: A promising method for the diagnosis of onychomycosis. An ex vivo pilot study.

Trichophyton rubrum and Candida species comprise the majority of onychomycosis pathogens. The aim of this study was to evaluate Raman spectroscopy for the differentiation between healthy and either T. rubrum or Candida infected nails. Raman measurements were performed on clippings (N = 52) infected either by T. rubrum (N = 12) or Candida species (N = 14; C. parapsilosis (sensu lato): N = 11, C. glabrata: N = 1, C. albicans: N = 2) with healthy nails (N = 26) used as controls. Systematic spectral differences were observed in the 500-520 cm-1 band region, attributable to a diverting imprint of the disulfide stretching of cystine and cysteine residues among samples. Particularly, Candida infected nails demonstrated a shoulder at 519 cm-1, corresponding to the signal of the less stable gauche-gauche-trans conformation of the disulfide bond. Two additional bands at 619 and 648 cm-1, corresponding to the C-S stretching vibration, were more evident in the T. rubrum infected nails. Finally, a Raman band at 1550 cm-1, attributable to amide II and tryptophan (Trp) content, was undetectable in Candida infected nails. Using principal component analysis (PCA), efficient differentiation of healthy, T. rubrum and Candida species infected nails was achieved. Soft independent modeling of class analogy (SIMCA) and partial least squares-discriminant analysis (PLS-DA) were further applied to generate diagnostic algorithms for the classification of Raman spectra. Both techniques succeeded in modeling clinical nail samples in three groups according to their mycological categories. Raman spectroscopy is a promising method for the differentiation of healthy vs. diseased nails, including efficient differentiation between onychomycosis caused by T. rubrum and Candida species.

High variability within Candida albicans transcription factor RLM1: Isolates from vulvovaginal infections show a clear bias toward high molecular weight alleles.

Previous studies have correlated the severity of recurrent vulvovaginal Candida infections (VVC) and balanitis in patients from China with the presence of some dominant genotypes at the ORF RLM1. Here we tested VVC vs non-VVC isolates from Portugal, Brazil and Greece and, although the same genotypes were identified in VVC isolates, they were present in only five out of 150 strains. However, this analysis showed that VVC isolates presented a higher percentage of genotypes with similar high molecular weight alleles, in comparison with strains isolated from other biological sources.

Fundamental niche prediction of the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii in Europe.

Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe and Mediterranean area.

Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population.

Cryptococcus neoformans var. neoformans (serotype D) represents about 30% of the clinical isolates in Europe and is present less frequently in the other continents. It is the prevalent etiological agent in primary cutaneous cryptococcosis as well as in cryptococcal skin lesions of disseminated cryptococcosis. Very little is known about the genotypic diversity of this Cryptococcus subtype. The aim of this study was to investigate the genotypic diversity among a set of clinical and environmental C. neoformans var. neoformans isolates and to evaluate the relationship between genotypes, geographical origin and clinical manifestations. A total of 83 globally collected C. neoformans var. neoformans isolates from Italy, Germany, France, Belgium, Denmark, Greece, Turkey, Thailand, Japan, Colombia, and the USA, recovered from different sources (primary and secondary cutaneous cryptococcosis, disseminated cryptococcosis, the environment, and animals), were included in the study. All isolates were confirmed to belong to genotype VNIV by molecular typing and they were further investigated by MLST analysis. Maximum likelihood phylogenetic as well as network analysis strongly suggested the existence of a recombinant rather than a clonal population structure. Geographical origin and source of isolation were not correlated with a specific MLST genotype. The comparison with a set of outgroup C. neoformans var. grubii isolates provided clear evidence that the two varieties have different population structures.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin.

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

Epizootic and epidemic dermatophytose outbreaks caused by Trichophyton mentagrophytes from rabbits in Portugal, 2015.

We report an outbreak of dermatophytoses in rabbits, which was the origin of a dermatophytose epidemic in an agricultural school in central Portugal, affecting 15 people. Both the phenotypic characteristics and internal transcribed spacer (ITS) sequence of the dermatophytes isolated from the rabbits and patients were identical, suggesting that a single strain was responsible for both the epizootic and epidemic dermatophytoses and confirming that these two outbreaks were linked. The ITS sequences were also 100% identical to the ITS sequence of five strains isolated from rabbits in Greece and Italy, but different from that of Trichophyton mentagrophytes commonly isolated from dogs and cats. These results suggest that a particular T. mentagrophytes genotype could be prevalent in rabbits in southern Europe.

Pityriazepin and other potent AhR ligands isolated from Malassezia furfur yeast.

Malassezia furfur yeast strains isolated from diseased human skin preferentially biosynthesize indole alkaloids which can be detected in the human skin and are highly potent activators of the aryl hydrocarbon receptor (AhR) and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on l-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assay in recombinant cell lines derived from four different species, although significant species differences in relative potency were observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. M. furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner.

Caspofungin at catheter lock concentrations eradicates mature biofilms of Candida lusitaniae and Candida guilliermondii.

The antibiofilm activities of caspofungin, anidulafungin, micafungin, and liposomal amphotericin B were studied against Candida lusitaniae, Candida guilliermondii, and a Candida albicans control strain. While anidulafungin and micafungin (0.007 to 2,048 mg/liter) showed reduced activity against biofilms of both test species, caspofungin displayed concentration-dependent antibiofilm activity, reaching complete and persistent eradication at concentrations achievable during lock therapy (512 to 2,048 mg/liter, P < 0.05). Although liposomal amphotericin B strongly inhibited mature biofilms, it possessed lower antibiofilm activity than caspofungin (P < 0.05).

The Malassezia genus in skin and systemic diseases.

In the last 15 years, the genus Malassezia has been a topic of intense basic research on taxonomy, physiology, biochemistry, ecology, immunology, and metabolomics. Currently, the genus encompasses 14 species. The 1996 revision of the genus resulted in seven accepted taxa: M. furfur, M. pachydermatis, M. sympodialis, M. globosa, M. obtusa, M. restricta, and M. slooffiae. In the last decade, seven new taxa isolated from healthy and lesional human and animal skin have been accepted: M. dermatis, M. japonica, M. yamatoensis, M. nana, M. caprae, M. equina, and M. cuniculi. However, forthcoming multidisciplinary research is expected to show the etiopathological relationships between these new species and skin diseases. Hitherto, basic and clinical research has established etiological links between Malassezia yeasts, pityriasis versicolor, and sepsis of neonates and immunocompromised individuals. Their role in aggravating seborrheic dermatitis, dandruff, folliculitis, and onychomycosis, though often supported by histopathological evidence and favorable antifungal therapeutic outcomes, remains under investigation. A close association between skin and Malassezia IgE binding allergens in atopic eczema has been shown, while laboratory data support a role in psoriasis exacerbations. Finally, metabolomic research resulted in the proposal of a hypothesis on the contribution of Malassezia-synthesized aryl hydrocarbon receptor (AhR) ligands to basal cell carcinoma through UV radiation-induced carcinogenesis.

Species-specific and drug-specific differences in susceptibility of Candida biofilms to echinocandins: characterization of less common bloodstream isolates.

Candida species other than Candida albicans are increasingly recognized as causes of biofilm-associated infections. This is a comprehensive study that compared the in vitro activities of all three echinocandins against biofilms formed by different common and infrequently identified Candida isolates. We determined the activities of anidulafungin (ANID), caspofungin (CAS), and micafungin (MFG) against planktonic cells and biofilms of bloodstream isolates of C. albicans (15 strains), Candida parapsilosis (6 strains), Candida lusitaniae (16 strains), Candida guilliermondii (5 strains), and Candida krusei (12 strains) by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. Planktonic and biofilm MICs were defined as ≥ 50% fungal damage. Planktonic cells of all Candida species were susceptible to the three echinocandins, with MICs of ≤ 1 mg/liter. By comparison, differences in the MIC profiles of biofilms in response to echinocandins existed among the Candida species. Thus, C. lusitaniae and C. guilliermondii biofilms were highly recalcitrant to all echinocandins, with MICs of ≥ 32 mg/liter. In contrast, the MICs of all three echinocandins for C. albicans and C. krusei biofilms were relatively low (MICs ≤ 1 mg/liter). While echinocandins exhibited generally high MICs against C. parapsilosis biofilms, MFG exhibited the lowest MICs against these isolates (4 mg/liter). A paradoxical growth effect was observed with CAS concentrations ranging from 8 to 64 mg/liter against C. albicans and C. parapsilosis biofilms but not against C. krusei, C. lusitaniae, or C. guilliermondii. While non-albicans Candida planktonic cells were susceptible to all echinocandins, there were drug- and species-specific differences in susceptibility among biofilms of the various Candida species, with C. lusitaniae and C. guilliermondii exhibiting profiles of high MICs of the three echinocandins.

Single-dose pharmacodynamics of amphotericin B against Aspergillus species in an in vitro pharmacokinetic/pharmacodynamic model.

Conventional MIC testing of amphotericin B results in narrow MIC ranges challenging the detection of resistant strains. In order to discern amphotericin B pharmacodynamics, the in vitro activity of amphotericin B was studied against Aspergillus isolates with the same MICs by using a new in vitro pharmacokinetic/pharmacodynamic (PK/PD) model that simulates amphotericin B human plasma levels. Clinical isolates of Aspergillus fumigatus, A. terreus, and A. flavus with the same Clinical and Laboratory Standards Institute modal MICs of 1 mg/liter were exposed to amphotericin B concentrations following the plasma concentration-time profile after single-bolus administration with C(max) values of 0.6, 1.2, 2.4, and 4.8 mg/liter. Fungal growth was monitored for up to 72 h based on galactomannan production. Complete growth inhibition was observed only against A. fumigatus with amphotericin B with a Cmax of ≥ 2.4 mg/liter. At the lower C(max) values 0.6 and 1.2 mg/liter, significant growth delays of 34 and 52 h were observed, respectively (P < 0.001). For A. flavus, there was no complete inhibition but a progressive growth delay of 1 to 50 h at an amphotericin B C(max) of 0.6 to 4.8 mg/liter (P < 0.001). For A. terreus, the growth delay was modest (up to 8 h) at all C(max)s (P < 0.05). The C(max) (95% confidence interval) associated with 50% activity for A. fumigatus was 0.60 (0.49 to 0.72) mg/liter, which was significantly lower than for A. flavus 3.06 (2.46 to 3.80) mg/liter and for A. terreus 7.90 (5.20 to 12.29) mg/liter (P < 0.001). A differential in vitro activity of amphotericin B was found among Aspergillus species despite the same MIC in the order A. fumigatus > A. flavus > A. terreus in the in vitro PK/PD model, possibly reflecting the different concentration- and time-dependent inhibitory/killing activities amphotericin B exerted against these species.

Identification of medically relevant species of arthroconidial yeasts by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used for an extensive identification study of arthroconidial yeasts, using 85 reference strains from the CBS-KNAW yeast collection and 134 clinical isolates collected from medical centers in Qatar, Greece, and Romania. The test set included 72 strains of ascomycetous yeasts (Galactomyces, Geotrichum, Saprochaete, and Magnusiomyces spp.) and 147 strains of basidiomycetous yeasts (Trichosporon and Guehomyces spp.). With minimal preparation time, MALDI-TOF MS proved to be an excellent diagnostic tool that provided reliable identification of most (98%) of the tested strains to the species level, with good discriminatory power. The majority of strains were correctly identified at the species level with good scores (>2.0) and seven of the tested strains with log score values between 1.7 and 2.0. The MALDI-TOF MS results obtained were consistent with validated internal transcribed spacer (ITS) and/or large subunit (LSU) ribosomal DNA sequencing results. Expanding the mass spectrum database by increasing the number of reference strains for closely related species, including those of nonclinical origin, should enhance the usefulness of MALDI-TOF MS-based diagnostic analysis of these arthroconidial fungi in medical and other laboratories.

Sexual reproduction in the opportunistic human pathogen Aspergillus terreus.

Aspergillus terreus has a worldwide distribution in soil, constitutes the third most important cause of invasive aspergillosis in humans and is classically regarded as a strictly asexual species. Strains of A. terreus were characterized by ITS, ß-tubulin and calmodulin sequences. Mating type was identified by amplifying and sequencing MAT1-1 and MAT1-2 genes. One MAT1-2 strain produced hyphal masses when crossed with four MAT1-1 strains on mixed cereal agar after 3 wk at 37 C. Cleistothecia formed inside the hyphal masses and produced asci containing smooth-walled ascospores with an equatorial protuberance. This is the first report of sexual reproduction in A. terreus.